6FX7

Crystal structure of in vitro evolved Af1521

6FX7 の概要

• エントリーDOI: 10.2210/pdb6fx7/pdb
• 分子名称: [Protein ADP-ribosylglutamate] hydrolase AF_1521, [(2R,3S,4R,5R)-5-(6-AMINOPURIN-9-YL)-3,4-DIHYDROXY-OXOLAN-2-YL]METHYL [HYDROXY-[[(2R,3S,4R,5S)-3,4,5-TRIHYDROXYOXOLAN-2-YL]METHOXY]PHOSPHORYL] HYDROGEN PHOSPHATE (3 entities in total)
• 機能のキーワード: macro domain, adp-ribose binding, signaling protein
• 由来する生物種: Archaeoglobus fulgidus DSM 4304
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 24135.26

構造登録者

Karlberg, T.,Thorsell, A.G.,Nowak, K.,Hottiger, M.O.,Schuler, H. (登録日: 2018-03-08, 公開日: 2019-09-25, 最終更新日: 2024-11-06)

主引用文献

Nowak, K.,Rosenthal, F.,Karlberg, T.,Butepage, M.,Thorsell, A.G.,Dreier, B.,Grossmann, J.,Sobek, J.,Imhof, R.,Luscher, B.,Schuler, H.,Pluckthun, A.,Leslie Pedrioli, D.M.,Hottiger, M.O.
Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.
Nat Commun, 11:5199-5199, 2020

PubMed Abstract: Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

PubMed: 33060572
DOI: 10.1038/s41467-020-18981-w

実験手法

X-RAY DIFFRACTION (1.82 Å)